It is conventional practice in plant breeding or plant advancement experiments to grow plants from seed of known parentage. The seed are planted in experimental plots, growth chambers, greenhouses, or other growing conditions in which they are either cross pollinated with other plants of known parentage or self pollinated. The resulting seed are the offspring of the two parent plants or the self pollinated plant, and are harvested, processed and planted to continue the plant breeding cycle. Specific laboratory or field-based tests may be performed on the plants, plant tissues, seed or seed tissues, in order to aid in the breeding or advancement selection process.
Generations of plants based on known crosses or self pollinations are planted and then tested to see if these lines or varieties are moving towards characteristics that are desirable in the marketplace. Examples of desirable traits include, but are not limited to, increased yield, increased homozygosity, improved or newly conferred resistance and/or tolerance to specific herbicides and/or pests and pathogens, increased oil content, altered starch content, nutraceutical composition, drought tolerance, and specific morphological based trait enhancements.
As can be appreciated and as is well known in the art, these experiments can be massive in scale. They involve a huge labor force ranging from scientists to field staff to design, plant, maintain, and conduct the experiments, which can involve thousands or tens of thousands of individual plants. They also require substantial land resources. Plots or greenhouses can take up thousands of acres of land. Not only does this tie up large amounts of land for months while the plants germinate, grow, and produce seed, during which time they may be sampled for laboratory or field testing, but then the massive amounts of seed must be individually tagged, harvested and processed.
A further complication is that much of the experimentation goes for naught. It has been reported in literature that some seed companies discard 80-90% of the plants in any generation early on in the experiment. Thus, much of the land, labor and material resources expended for growing, harvesting, and post-harvest processing ultimately are wasted for a large percentage of the seed.
Timing pressures are also a factor. Significant advances in plant breeding have put more pressure on seed companies to more quickly advance lines or varieties of plants for more and better traits and characteristics. The plant breeders and associated workers are thus under increasing pressure to more efficiently and effectively process these generations and to make more and earlier selections of plants which should be continued into the next generation of breeding.
Therefore, a movement towards earlier identification of traits of interest through laboratory based seed testing has emerged. Seed is non-destructively tested to derive genotypic information. If traits of interest are identified, the selected seed from specific plants are used either for further experiments and advancement, or to produce commercial quantities. Testing seed prevents the need to grow the seed into immature plants, which are then tested. This saves time, space, and effort. Effective, early identification of desirable traits in seed can lead to greatly reducing the amount of land needed for experimental testing, the amount of seed that must be tested, and the amount of time needed to derive the information needed to advance the experiments. For example, instead of thousands of acres of plantings and the subsequent handling and processing of all those plants, a fraction of acres and plants might be enough. However, because timing is still important, this is still a substantial task because even such a reduction involves processing, for example, thousands of seed per day.
A conventional method of attempting non-lethal seed sampling is as follows. A single seed of interest is held with pliers above a sheet of paper laid out on a surface. A small drill bit is used to drill into a small location on the seed. Debris removed by the drill bit from the seed is collected on the sheet of paper. The paper is lifted and the debris is transferred to a test tube or other container. The debris is thus collected and ready for laboratory analysis. The seed is stored in another container. The two containers, housing the seed and sample, are indexed or correlated for tracking purposes. This method is intended to be non-lethal to the seed. However, the process is slow. Its success and effectiveness depends heavily on the attention and accuracy of the worker. Each single seed must be manually picked up and held by the pliers. The drilling is also manual. Care must be taken with the drilling and the handling of the debris, as well as insuring that the full sample amount is transferred into a container and the seed from which the sample was taken into another container. These two containers, e.g. the individual test tubes, must then be handled and marked or otherwise tracked and identified. Additionally, the pliers and drill must be cleaned between the sampling of each seed. There can be substantial risk of contamination by carry-over from sample to sample and the manual handling. Also, many times it is desirable to obtain seed material from a certain physiological tissue of the seed. For example, with corn seed, it may be desirable to take the sample from the endosperm. In such cases, it is not trivial, but rather time-consuming and somewhat difficult to manually grasp a small corn seed is such a way to allow the endosperm to be oriented to expose it for drilling. Sampling from other seed structures such as the seed germ must be avoided because sampling from such regions of the seed negatively impacts germination rates. Sometimes it is difficult to obtain a useful amount of sample with this method. In summary, sampling from seed relies heavily on the skill of the worker and is relative to throughput and accuracy, including whether the procedure gives the seed a good chance at germination. These issues are amplified when a worker is charged with processing many seed a day.
As evidenced by these examples, present conventional seed analysis methods, such as is used in genotypic analysis, require at least a part of the seed to be removed and processed. In removing a portion of the seed, various objectives may need to be met. These may include one or more of the following objectives:
(a) maintain seed viability post-sampling if required;
(b) obtain at least a minimum required sample amount, without affecting viability;
(c) obtain a sample from a specific location on the seed, often requiring the ability to efficiently position and orient the seed in a specific position and orientation for sampling;
(d) maintain a particular throughput level for efficiency purposes;
(e) reduce or virtually eliminate contamination between samples;
(f) maintain an efficient and controlled post-sampling handling regimen and environment to move and collect seed portion and seed after sampling; and
(g) allow for the tracking of separate samples and their correlation to other samples in a group.
(a) Viability
With regard to maintaining seed viability, it may be critical in some circumstances that the seed sampling method and apparatus not damage the seed in such a way that seed viability is reduced. It is often desirable that such analysis be non-lethal to the seed, or at least result in a substantial probability that the sampled seed will germinate (e.g. no significant decrease in germination potential) so that it can be grown into a mature plant. For some analyses, seed viability does not need to be maintained, in which case larger samples can often be taken. The need for seed viability will depend on the intended use of the seeds post-sampling. Therefore, there is a need to preserve the viability of the seed by providing seed sampling and handling apparatus, methods and systems of the present invention.
(b) Sample Amount
It is desirable to obtain a useful amount of sample. To be useful, in some applications it must be above a certain minimum amount necessary in order to perform a given test and obtain a meaningful result. Different tests or assays require different sample amounts. It may be equally important to avoid taking too much tissue for a sample, because a sample that is too large may reduce germination potential of a seed, which may be undesirable. Therefore, it is desirable that sampling apparatus, methods and systems allow for variation in the amount of sample taken from any given seed.
(c) Sample Location
A useful sample amount also can involve sample location accuracy. For example, in some applications the sample must come only from a certain location or from certain tissue. Further, it is difficult to handle small seed. It is also difficult to accurately position and orient seed. On a corn seed, for example, it may be important to sample the endosperm tissue, and orient the corn seed for sampling that particular tissue. Therefore, it is desirable that the sampling apparatus, methods and systems are adapted to allow for high throughput seed positioning and orientation of seed for location-specific sampling, which may include seed orientation apparatuses, methods and systems with geometries, architecture and steps adapted to position and orient seed in a predetermined orientation.
(d) Throughput
A sampling apparatus and methodology must consider the throughput level that supports the required number of samples being taken in a time efficient manner. For example, some situations involve the potential need to sample thousands, hundreds of thousands, or even millions of seed per year. Taking the hypothetical example of a million seed per year, and a 5-day work week, this would average nearly four thousand samples per day for each working day of a year. It is difficult to meet such demand with lower throughput sampling methods. Accordingly, higher throughput, automatic or even semi-automatic apparatuses, methods and systems are desirable.
(e) Avoiding Contamination
It is desirable that a sampling methodology, system and apparatus not be prone to cross-contamination in order to maintain sample purities for subsequent analytical testing procedures. This can involve not only sample location accuracy, such that a sample from a given location is not contaminated with tissue from a different location, but also the method of sampling and the handling of each individual sample, ensuring no contamination between samples.
(f) Handling (Post-Sampling)
With higher throughput as an objective, it is important that consideration be given to maintaining an efficient and controlled post-sampling handling regimen and environment to move and collect the seed portion and seed after sampling. Such post-sampling operations should ensure each operation is devoid of contamination. Depending on the tool used to remove a portion of the seed, such as a laser, further consideration need to be given to how the seed and seed portion are handled and collected to insure viability is preserved, contamination is limited, and accurate high throughput separation of seed and seed portions is maintained.
(g) Indexing (Tracking) Sample and Sampled Seed
Efficient processing of seed and samples removed from seed presents a variety of issues and challenges, especially when it is important to keep track of each seed, each sample, and their correlation to each other, or to other samples. Accordingly, it is desirable that sampling apparatus, methods and systems allow for easy tracking and correlation of seed and their samples.
Conventional seed sampling technologies do not address these requirements sufficiently, resulting in pressures on capital and labor resources, and thus illustrate the need for an improvement in the state of the art. The current apparatuses, methods and systems are relatively low throughput, have substantial risk of cross-contamination, and tend to be inconsistent because of a reliance on significant manual handling, orienting, removal, post-handling, tracking and correlation of the sample and the seed. This can affect the type of sample taken from the seed and the likelihood that the seed will germinate. There is a need to eliminate the resources current methods require for cleaning between samples. There is a need to reduce or minimize cross-contamination between samples by carry-over or other reasons, or any contamination from any source of any sample. There is also a need for more reliability and accuracy. There is a further need to provide high throughput handling means for the seed and seed part. Accordingly, there is a need for methodologies and systems and their corresponding apparatuses which provide for seed sampling that accomplishes one or more of the following objectives:
(a) maintain seed viability post-sampling if required;
(b) obtain at least a minimum required sample amount, without affecting viability;
(c) obtain a sample from a specific location on the seed, often requiring the ability to efficiently position and orient the seed in a specific position and orientation for sampling;
(d) maintain a particular throughput level for efficiency purposes;
(e) reduce or virtually eliminate contamination between samples;
(f) maintain an efficient, high throughput and controlled post-sampling handling regimen and environment to move and collect the seed portion and seed after sampling; and
(g) allow for the tracking of separate seed parts and their correlation to other samples in a group.
Some of these objectives that are desirable when sampling seed can be conflicting and even antagonistic. For example, high throughput methodologies may require relatively rapid operation but with relatively high accuracy and low contamination risk, such that they must be done more slowly than is technically possible. These multiple objectives have therefore existed in the art and have not been satisfactorily addressed or balanced by the currently available apparatuses, methods and systems. There is a need in the art to overcome the above-described types of problems such that the maximum number of objectives is realized in any given embodiment.